Tissue injury and inflammation lead to the development of an evident facilitation in the sensitivity to moderately aversive stimuli, e.g. hyperalgesia. It has been long appreciated that this phenomenon is diminished by agents that block cyclooxygenase (COX) activity (Vane, Nat. New Biol., 231:232-235, 1971). While early work suggested that this action resulted from a peripheral effect (Ferreira, Nat. New Biol., 240:200-203, 1972), it was subsequently found that inhibition of spinal COX also led to reversal of the facilitated state (Yaksh, et al., “Acetylsalicilic Acid: New Uses for an Old Drug”, pp. 137-152 (Barnet, et al., editors) Raven Press, 1982; Taiwo and Levine, J. Neurosci., 8:1346-1349, 1988). Consistent with this action, persistent small afferent input, as arises from tissue injury, was shown to evoke a significant spinal release of prostanoids in vivo in a mariner that was blocked by spinally-delivered COX inhibitors (Ramwell, et al., Am. J. Physiol., 211:998-1004, 1966; Yaksh, supra, 1982; Malmberg and Yaksh, Science, 257:1276-1279, 1992; Malmberg and Yaksh, J. Neurosci., 15:2768-2776, 1995; Ebersberger, et al., 1999, Samad et al., Nature, 410:471-475, 2001, and Yaksh, et al., J. Neurosci., 21:5847-5853, 2001). An important element of prostaglandin (PG) synthesis is phospholipase A2 (PLA2), as it is required to generate arachidonic acid, which is the substrate for COX-mediated prostanoid formation.
Phospholipase A2 (PLA2) constitutes a super-family of enzymes that catalyze the hydrolysis of the fatty acid ester from the sn-2 position of membrane phospholipids, yielding a free fatty acid and a lysophospholipid. Among the intracellular PLA2, are the cytosolic Group IVA PLA2 (GIVA PLA2, also referred to herein as cPLA2), which is generally considered a pro-inflammatory enzyme; the calcium-independent Group VIA PLA2 (GVIA PLA2, also referred to herein as iPLA2); and, secreted Group V PLA2 (sPLA2). GVIA PLA2 is actually a group of cytosolic enzymes ranging from 85 to 88 kDa and expressed as several distinct splice variants of the same gene, only two of which have been shown to be catalytically active (Group VIA-1 and VIA-2 PLA2, see Larsson, et al., J. Biol. Chem. 273: 207-214, 1998). The role of GVIA PLA2 in the inflammatory process is unclear, but this enzyme appears to be the primary PLA2 for basal metabolic functions within the cell, reportedly including membrane homeostasis (Balsinde, et al., Proc. Natl. Acad. Sci. U.S.A., 92:8527-8531, 1995; Balsinde, et al., J. Biol. Chem., 272: 29317-29321, 1997; Balsinde, et al., J. Biol. Chem., 272:16069-16072, 1997; Ramanadham, et al., J. Biol. Chem., 274:13915-13927, 1999; Birbes, et al., Eur. J. Biochem., 267:7118-7127, 2000; and Ma, et al., Lipids, 36:689-700, 2001.), insulin receptor signaling (Ramanadham, et al., J. Biol. Chem., 274: 13915-13927, 1999; Ma, et al., J. Biol. Chem., 276: 13198-13208, 2001) and calcium channel regulation (Guo, et al., J. Biol. Chem., 277: 32807-32814, 2002; Cummings, et al., Am. J. Physiol. Renal Physiol., 283: F492-498, 2002). GVIA, GIVA and GV PLA2 are all present and play active roles in central nervous system inflammatory processes (see, e.g., Sun, et al., J. Lipid Res., 45:205-213, 2004).
The GVIA PLA2 enzymes all contain a consensus lipase motif, Gly-Thr-Ser*-Thr-Gly, with the catalytic serine confirmed by site-directed mutagenesis (Larsson, et al., J. Biol. Chem., 273:207-14, 1998; Tang, et al., J. Biol. Chem., 272: 8567-8575, 2002). Other residues critical for catalysis have yet to be confirmed, and while the mechanism by which it cleaves the sn-2 linkage has not been established, GVIA PLA2 is likely to be an hydrolase with a catalytic Ser/Asp dyad similar to Group IVA PLA2 (Dessen, et al., Cell 1999, 97: 349-360, 1999; Dessen, Biochim. Biophys. Acta, 1488:40-47, 2000; Phillips, et al., J. Biol. Chem., 278: 41326-41332, 2003). Constitutive mRNA and protein have been detected in the spinal cord for group IVA calcium-dependent PLA2 (Group IVA cPLA2) and Group VIA calcium-independent iPLA2 (Group VIA iPLA2) and secretory Group II and V sPLA2 forms (Lucas, et al., Br. J. Pharmacol., 144:940-952, 2005, Svensson et al., Annu. Rev. Pharmacol. Toxicol., 42:553-555, 2005).
The discovery of a novel structural series of 2-oxoamides that inhibit Group IVA cPLA2 in vitro and in vivo (Kokotos, et al., J. Med. Chem., 45:2891-2893, 2002; Kokotos, et al., J. Med. Chem., 47:3615-3628, 2004) was recently reported. In that initial work, 2-oxoamides were observed to inhibit inflammation in the rat paw carrageenan-induced edema assay (Kokotos, et al., supra, 2004). Based upon the similarity of substrates, classes of common inhibitors, very limited sequence homology in the region of the catalytic serine, and similarities in the active sites of GIVA and GVIA PLA2, GIVA PLA2 may show cross-reactivity with GVIA PLA2. It has been difficult, however, to design selective inhibitors that can distinguish between these active sites in vivo. Thus, what is need in the art are selective Group IVA cPLA2 in vitro and in vivo inhibitors.